P1334: THE IMPACT OF SMALL MOLECULE INHIBITION OF METTL3 ON NORMAL HAEMATOPOIESIS
Authors: Katherine Sturgess, Ms Vijayabaskar, Eliza Yankova, Justyna Rak, Maria Eleftheriou, Tomoya Isobe, Melania Barile, Malgorzata Gozdecka, Berthold Gottgens, Nicola Wilson, Kostas Tzelepis
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Targeting RNA methylation by selective catalytic inhibition of the RNA methyltransferase METTL3 has shown significant promise as a therapeutic strategy against AML. While hematopoietic defects have been reported in METTL3 KO models, non-catalytic functions of METTL3 are increasingly being recognized, highlighting the importance of identifying the specific effects of its catalytic inhibition. Here we report the first characterization of the effects of METTL3 catalytic inhibition on healthy murine hematopoiesis. Aims: To elucidate the effects of METTL3 catalytic inhibition with STM2457 on healthy hematopoiesis. Methods: We performed 10X Genomics scRNA-seq of hematopoietic stem and progenitor cells (HSPCs) from 3 STM2457-treated mice and 4 vehicle-treated controls, with FACS analysis of peripheral blood from the same cohort of animals. Results: Consistent with previous reports, flow cytometric sorting demonstrated expansion of the HSPC compartment of STM2457-treated mice, with increased numbers of lineage-c-kit+ Sca1+ (LSK) and LSK CD150+CD48- HSCs. Differential cell abundance analysis in the scRNA-seq data indicated a significant depletion of erythroid progenitors, with an increase in myeloid progenitors and no significant change in the abundance of HSCs (FDR <0.2). The discordance between phenotypic and transcriptional HSC abundance likely reflects upregulation of phenotypic markers including Sca1 and CD150 in the inhibitor-treated bone marrow. Lineage probability analysis was performed to infer cell fate potential, demonstrating a lineage bias towards myeloid and away from erythroid differentiation, which was detectable even in the transcriptionally defined HSC cluster. Differential gene expression analysis revealed enrichment across the whole STM2457-treated hematopoietic landscape for inflammatory and interferon response genes, consistent with aberrant stabilization of endogenous RNAs stimulating the dsRNA pattern recognition response. There was a global increase in quiescence in the STM2457-treated bone marrow. Genes associated with emergency granulopoiesis and regulators of HSPC quiescence were among the most significantly upregulated. Trajectory-based differential expression analysis uncovered lineage- and stage-specific dynamic gene expression changes in the neutrophil and erythroid trajectories. Along the erythroid trajectory, prolonged upregulation of Ccnd2 may contribute to increased quiescence. Downregulation of Ccnd2 and consequent shortening of the G1 phase of cell cycle has been reported to govern differentiation of committed erythroid progenitors, and failure to progress into S phase prevents terminal erythroid differentiation. Taken together, these results suggest a dsRNA-driven inflammatory response primes the earliest progenitors towards a tissue-level lineage bias. Summary/Conclusion: Here we report the first characterization of the impact of METTL3 catalytic inhibition by STM2457 on healthy adult murine hematopoiesis. A compartment-wide inflammatory transcriptional signature is consistent with effective N6-methyladenosine (m6A) depletion causing dsRNA formation. While phenotypic HSPCs are expanded there is no significant impact on the number of transcriptionally-defined HSCs. A significant erythroid defect is associated with increased quiescence in early erythroid progenitors. Collectively, these results show that targeted inhibition of METTL3 methyltransferase activity produces a milder and more specific hematopoietic phenotype than previously described in knock-out animals, with no significant impact on mature peripheral blood cell counts. Keywords: Methylation, Hematopoiesis